Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

ABSTRACT

The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a division of U.S. patent application Ser.No. 14/019,911, filed 6 Sep. 2013, which in turn is a continuation ofU.S. patent application Ser. No. 13/178,844, filed 8 Jul. 2011, which inturn is a continuation of U.S. patent application Ser. No. 12/855,223,filed 12 Aug. 2010, which in turn is a continuation of U.S. patentapplication Ser. No. 11/079,476 filed 15 Mar. 2005, which in turn isrelated to and claims priority under 35 U.S.C. § 119(e) to U.S.provisional patent application No. 60/553,487 filed 15 Mar. 2004. Eachapplication is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made in part with Government support under GrantNumbers AI29329 and HL074704 awarded by the National Institute ofHealth. The Government has certain rights in this invention.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1954580SequenceListing.txt, was created on 13 Jun. 2016 and is 17 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention pertains to compositions and methods for gene-specificinhibition of gene expression by double-stranded ribonucleic acid(dsRNA) effector molecules. The compositions and methods are useful inmodulating gene expression in a variety of applications, includingtherapeutic, diagnostic, target validation, and genomic discovery.

BACKGROUND OF THE INVENTION

Suppression gene expression by double-stranded RNA (dsRNA) has beendemonstrated in a variety of systems including plants(post-transcriptional gene suppression) (Napoli et al., 1990, PlantCell. 2:279-289), fungi (quelling) (Romano and Marcino, 1992, MolMicrobiol. 6:3343-53), and nematodes (RNA interference) (Fire et al.,1998, Nature 391:806-811). Early attempts to similarly suppress geneexpression using long dsRNAs in mammalians systems failed due toactivation of interferon pathways that do not exist in lower organisms.Interferon responses are triggered by dsRNAs (Stark et al., 1998, Annu.Rev. Biochem., 67:227-264). In particular, the protein kinase PKR isactivated by dsRNAs of greater than 30 bp long (Manche et al., 1992, MolCell Biol., 12:5238-48) and results in phosphorylation of translationinitiation factor eIF2c which leads to arrest of protein synthesis andactivation of 2′5′-oligoadenylate synthetase (2′-5′-OAS), which leads toRNA degradation (Minks et al., 1979, J. Biol. Chem. 254:10180-10183).

In Drosophila cells and cell extracts, dsRNAs of 150 bp length orgreater were seen to induce RNA interference while shorter dsRNAs wereineffective (Tuschl et al., 1999, Genes & Dev., 13:3191-3197). Longdouble-stranded RNA, however, is not the active effecter molecule; longdsRNAs are degraded by an RNase III class enzyme called Dicer (Bernsteinet al., 2001, Nature, 409:363-366) into very short 21-23 bp duplexesthat have 2-base 3′-overhangs (Zamore et al., 2000, Cell, 101:25-33).These short RNA duplexes, called siRNAs, direct the RNAi response invivo and transfection of short chemically synthesized siRNA duplexes ofthis design permits use of RNAi methods to suppress gene expression inmammalian cells without triggering unwanted interferon responses(Elbashir et al., 2001, Nature, 411:494-498). The antisense strand ofthe siRNA duplex serves as a sequence-specific guide that directsactivity of an endoribonuclease function in the RNA induced silencingcomplex (RISC) to degrade target mRNA (Martinez et al., 2002, Cell,110:563-574).

In studying the size limits for RNAi in Drosophila embryo extracts invitro, a lower threshold of around 38 bp double-stranded RNA wasestablished for activation of RNA interference using exogenouslysupplied double-stranded RNA and duplexes of 36, 30, and 29 bp lengthwere without effect (Elbashir et al., 2001, Genes & Dev., 15:188-200).The short 30-base RNAs were not cleaved into active 21-23-base siRNAsand therefore were deemed inactive for use in RNAi (Elbashir et al.,2001, Genes & Dev., 15:188-200). Continuing to work in the Drosophilaembryo extract system, the same group later carefully mapped thestructural features needed for short chemically synthesized RNA duplexesto function as siRNAs in RNAi pathways. RNA duplexes of 21-bp lengthwith 2-base 3′-overhangs were most effective, duplexes of 20, 22, and23-bp length had slightly decreased potency but did result in RNAimediated mRNA degradation, and 24 and 25-bp duplexes were inactive(Elbashir et al., 2001, EMBO J., 20:6877-6888).

Some of the conclusions of these earlier studies may be specific to theDrosophila system employed. Other investigators established that longersiRNAs can work in human cells. However, duplexes in the 21-23-bp rangehave been shown to be more active and have become the accepted design(Caplen et al., 2001, Proc. Natl. Acad. Sci. USA, 98:9742-9747).Essentially, chemically synthesized duplex RNAs that mimicked thenatural products that result from Dicer degradation of long duplex RNAswere identified to be the preferred compound for use in RNAi.Approaching this problem from the opposite direction, investigatorsstudying size limits for RNAi in C. elegans found that although amicroinjected 26-bp RNA duplex could function to suppress geneexpression, it required a 250-fold increase in concentration comparedwith an 81-bp duplex (Parrish et al., 2000, Mol. Cell, 6:1077-1087).

Despite the attention given to RNAi research recently, the field isstill in the early stages of development. Not all siRNA molecules arecapable of targeting the destruction of their complementary RNAs in acell. As a result, complex sets of rules have been developed fordesigning RNAi molecules that will be effective. Those having skill inthe art expect to test multiple siRNA molecules to find functionalcompositions. (Ji et al. 2003) Some artisans pool several siRNApreparations together to increase the chance of obtaining silencing in asingle study. (Ji et al. 2003) Such pools typically contain 20 nM of amixture of siRNA oligonucleotide duplexes or more (Ji et al. 2003),despite the fact that a siRNA molecule can work at concentrations of 1nM or less (Holen et al. 2002). This technique can lead to artifactscaused by interactions of the siRNA sequences with other cellular RNAs(“off target effects”). (Scherer et al. 2003) Off target effects canoccur when the RNAi oligonucleotides have homology to unintended targetsor when the RISC complex incorporates the unintended strand from andRNAi duplex. (Scherer et al. 2003) Generally, these effects tend to bemore pronounced when higher concentrations of RNAi duplexes are used.(Scherer et al. 2003)

In addition, the duration of the effect of an effective RNAi treatmentis limited to about 4 days (Holen et al. 2002). Thus, researchers mustcarry out siRNA experiments within 2-3 days of transfection with ansiRNA duplex or work with plasmid or viral expression vectors to obtainlonger term silencing.

Additional physical studies are needed to more completely characterizethe structural requirements of RNAi active oligonucleotide duplexes toidentify more potent and longer lasting compositions and/or methods thatsimplify site-selection difficulties. These studies should also includea detailed analysis of the interferon response. Ideally, such studieswill be useful in identifying new RNAi active compounds that are morepotent, that simplify the site selection process, and decrease “offtarget effects.”

The invention provides RNAi compositions with increased potency,duration of action, and decreased “off target effects” that do notactivate the interferon response and provides methods for their use. Inaddition, the compositions ease site selection criteria and provide aduration of action that is about twice as long as prior knowncompositions. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides improved compositions and methods for selectivelyreducing the expression of a gene product from a desired target gene ina eukaryotic cell, as well as for treating diseases caused by theexpression of the gene. The method involves introducing into theenvironment of a cell an amount of a double-stranded RNA (dsRNA) suchthat a sufficient portion of the dsRNA can enter the cytoplasm of thecell to cause a reduction in the expression of the target gene. ThedsRNA has a first oligonucleotide sequence that is between 26 and about30 nucleotides in length and a second oligonucleotide sequence thatanneals to the first sequence under biological conditions, such as theconditions found in the cytoplasm of a cell. In addition, a region ofone of the sequences of the dsRNA having a sequence length of from about19 to about 23 nucleotides is complementary to a nucleotide sequence ofthe RNA produced from the target gene. A dsRNA composition of theinvention is at least as active as any isolated 19, 20, 21, 22, or 23basepair sequence that is contained within it Pharmaceuticalcompositions containing the disclosed dsRNA compositions are alsocontemplated. The compositions and methods give a surprising increase inthe potency and duration of action of the RNAi effect. Although theinvention is not intended to be limited by the underlying theory onwhich it is believed to operate, it is thought that this increase inpotency and duration of action are caused by the fact the dsRNA servesas a substrate for Dicer which appears to facilitate incorporation ofone sequence from the dsRNA into the RISC complex that is directlyresponsible for destruction of the RNA from the target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a comparison of RNAi efficacy using several dsRNAshaving variable length and formats including a two nucleotide 3′overhang (+2), a two nucleotide 5′ overhang (−2), and blunt ends (+0).The sequences are disclosed in the Example 2. In each panel A-D 200 μgof reporter vector was co-transfected with the indicated concentrationof dsRNA. Each bar represents the average of three duplicateexperiments. In FIG. 1A, 50 nM of each dsRNA was used. In FIG. 1B, 1 nMof each dsRNA was used. In FIG. 1C, 200 μM of each dsRNA was used. InFIG. 1D, 50 μM of each dsRNA was used.

FIG. 2 shows an RNAi assay in 3T3 cells expressing endogenous EGFP. Theexperimental procedure is described in Example 2. Measurements were made4 days after treatment. The dose response curves for 21-mer duplex with2-base 3′-overhang (SEQ ID No. 6/7), 25-mer duplex with 2-base5′-overhang (SEQ ID Nos. 16/17), and blunt 27-mer duplex (SEQ ID Nos.30/31) are shown.

FIG. 3 shows RNAi assays of various 27-mer RNA duplex formats asoutlined in Example 2. Duplex 27+0UU (SEQ ID Nos. 30/31) was mostpotent.

FIG. 4 shows RNAi assays on HEK 293 cells that were either mocktransfected (negative control), transfected with 200 ng EGFP reporterplasmid alone (positive control), or reporter plasmid+RNA duplexes atvarying concentrations as described in Example 3.

FIG. 5 shows superior knockout of the HNRPH1 gene by a 27-mer of theinvention as compared to a 21-mer directed to the same target. Westernblots obtained from HEK 293 cells after transfection with EGFP-specificsiRNA (SEQ ID No. 6/7; C) (negative control) and an HNRPH1 specific21-mer siRNA duplex (SEQ ID Nos. 51/52; 21+2) at varying concentrations,or with an HNRPH1 specific 27-mer siRNA duplex (SEQ ID Nos. 53/54; 27+0)at varying concentrations, as described in Example 4.

FIG. 6 shows the reaction of Dicer with various length RNA duplexes asdescribed in Example 5. Dicer was able to digest 25-29-mers primarilyinto about a 21 basepair duplex but dd not digest the 21 nucleotide longtest duplex.

FIG. 7 shows the relative expression of EGFP after RNAi assays using a27-mer dsRNA versus shorter 21-mer siRNAs contained within the 27-mersequence as described in more detail in Example 6. As shown a bluntended 27-mer that covers a poor site for a 21 nucleotide RNAi caneffectively target that site.

FIG. 8 shows the results of RNAi assays after treatment by variouseffector dsRNA molecules and pools of molecules as set forth in Example6.

FIG. 9 shows the time course study of the duration of the RNAi effectwith various effector molecules as described in Example 7. The studyshows the duration of the RNAi effect is at least about twice as longwith the 27-mer dsRNA of the invention as with 21-mers. The “27+0 UU”sequences are set forth in SEQ ID NOs:28 and 29. The “Mut-16” sequencesare set forth in SEQ ID NOs:70 and 71. The “Mut-16,17” sequences are setforth in SEQ ID NOs:72 and 73. The “Mut-15,16,17” sequences are setforth in SEQ ID NOs:74 and 75.

FIG. 10 shows the images of cells in a time course study of the durationof the RNAi effect with various effector molecules as described inExample 7. The study shows the duration of the RNAi effect is at leastabout twice as long with the 27-mer dsRNA of the invention as with21-mers.

FIG. 11 shows that neither interferon alpha (FIG. 11A) or interferonbeta (FIG. 11B) are induced by the 27-mer dsRNA of the invention asdescribed in more detail in Example 8.

FIG. 12 shows the results of a PKR activation assay in which long dsRNAresulted in strong PKR activation (positive control) while all of theshort synthetic RNAs showed no evidence for PKR activation.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compositions that contain double strandedRNA (“dsRNA”), and methods for preparing them, that are capable ofreducing the expression of target genes in eukaryotic cells. One of thestrands of the dsRNA contains a region of nucleotide sequence that has alength that ranges from about 19 to about 23 nucleotides that can directthe destruction of the RNA transcribed from the target gene.

For purposes of the invention a suitable dsRNA contains oneoligonucleotide sequence, a first sequence, that is at least 25nucleotides in length and no longer than about 30 nucleotides. Morepreferably this sequence of RNA is between about 26 and 29 nucleotidesin length. Still more preferably this sequence is about 27 or 28nucleotides in length, 27 nucleotides is most preferred. The secondsequence of the dsRNA can be any sequence that anneals to the firstsequence under biological conditions, such as within the cytoplasm of aeukaryotic cell. Generally, the second oligonucleotide sequence willhave at least 19 complementary base pairs with the first oligonucleotidesequence, more typically the second oligonucleotides sequence will haveabout 21 or more complementary base pairs, and more preferably about 25or more complementary base pairs with the first oligonucleotidesequence. In a preferred embodiment the second sequence is the samelength as the first sequence.

In certain embodiments the double-stranded RNA structure the first andsecond oligonucleotide sequences exist on separate oligonucleotidestrands which can be and typically are chemically synthesized. Inpreferred embodiments both strands are between 26 and 30 nucleotides inlength. In one preferred embodiment both strands are 27 nucleotides inlength, are completely complementary and have blunt ends. The dsRNA canbe from a single RNA oligonucleotide that undergoes intramolecularannealing or, more typically, the first and second sequences exist onseparate RNA oligonucleotides.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be chemically linked outside their annealing region by chemicallinking groups. Many suitable chemical linking groups are known in theart and can be used. Suitable groups will not block Dicer activity onthe dsRNA and will not interfere with the directed destruction of theRNA transcribed from the target gene.

The first and second oligonucleotide sequences are not required to becompletely complimentary. In fact, it is preferred that the 3′-terminusof the sense strand contains one or more mismatches. It is morepreferred that two mismatches be incorporated at the 3′terminus. In amost preferred embodiment the dsRNA of the invention is a doublestranded RNA molecule containing two RNA oligonucleotides each of whichis 27 nucleotides in length and, when annealed to each other, have bluntends and a two nucleotide mismatch on the 3′-terminus of the sensestrand (the 5′-terminus of the antisense strand).

One feature of the dsRNA compositions of the invention is that they canserve as a substrate for Dicer. Typically, the dsRNA compositions ofthis invention will not have been treated with Dicer, other RNAses, orextracts that contain them. Such treatments could digest the dsRNA tolengths of less than 25 nucleotides that are no longer Dicer substrates.Several methods are known and can be used for determining whether adsRNA composition serves as a substrate for Dicer. For example, Diceractivity can be measured in vitro using the Recombinant Dicer Enzyme Kit(GTS, San Diego, Ca) according to the manufacturer's instructions. Diceractivity can be measured in vivo by treating cells with dsRNA andmaintaining them for 24 h before harvesting them and isolating theirRNA. RNa can be isolated using standard methods, such as with theRNeasy™ Kit (Qiagen) according to the manufacturer's instructions. Theisolated RNA can be separated on a 10% PAGE gel which is used to preparea standard RNA blot that can be probed with a suitable labeleddeoxyoligonucleotide, such as an oligonucleotide labeled with theStarfire™ Oligo Labeling System (Integrated DNA Technologies, Inc.,Coralville, Iowa).

It has been found empirically that these longer dsRNA species of from 25to about 30 nucleotides give unexpectedly improved results in terms ofincreased potency and increased duration of action over shorter priorart RNAi compositions. The dsRNA compositions of the invention are atleast as active as any isolated 23 nucleotide dsRNA sequence containedwithin them and in preferred embodiments more active. Without wishing tobe bound by the underlying theory of the invention, it is thought thatthe longer dsRNA species serve as a substrate for the enzyme Dicer inthe cytoplasm of a cell. In addition to cleaving the dsRNA of theinvention into shorter segments, Dicer is thought to facilitate theincorporation of a single-stranded cleavage product derived from thecleaved dsRNA into the RISC complex that is responsible for thedestruction of the cytoplasmic RNA derived from the target gene. Studieshave shown that the cleavability of a dsRNA species by Dicer correspondswith increased potency and duration of action of the dsRNA species.

Suitable dsRNA compositions of this invention do not induce apoptosis inthe cells in which they are used. Apoptosis or “programmed cell death,”includes any non-necrotic, cell-regulated form of cell death, as definedby criteria well established in the art. Cells undergoing apoptosis showcharacteristic morphological and biochemical features. Once the processis triggered, or the cells are committed to undergoing apoptosis,morphological and physiological changes include cell shrinkage,chromatin condensation, nuclear and cytoplasmic condensation, membraneblebbing, partitioning of cytoplasm and nucleus into membrane boundvesicles which contain ribosomes (apoptotic bodies), and DNA degradationinto a characteristic oligonucleosomal ladder composed of multiples of200 base pairs, leading eventually to cell death. In vivo, theseapoptotic bodies are rapidly recognized and phagocytized by eithermacrophages or adjacent epithelial cells. In vitro, the apoptotic bodiesas well as the remaining cell fragments ultimately swell and finallylyse. This terminal phase of in vitro cell death has been termed“secondary necrosis.”

The effect that a dsRNA has on a cell can depend upon the cell itself.In some circumstances a dsRNA could induce apoptosis or gene silencingin one cell type and not another. Thus, it is possible that a dsRNAcould be suitable for use in one cell and not another. To be considered“suitable” a dsRNA composition need not be suitable under all possiblecircumstances in which it might be used, rather it need only be suitableunder a particular set of circumstances.

Modifications can be included in the disclosed dsRNA so long as thedsRNA remains sufficiently chemically stable, does not induce apoptosis,does not substantially interrupt annealing of the first and secondstrands, and otherwise does not substantially interfere with thedirected destruction of the RNA transcribed from the target gene.Modifications can be incorporated in the 3′-terminal region, the5′-terminal region, in both the 3′-terminal and 5′-terminal region or insome instances throughout the sequence. With the restrictions notedabove in mind any number and combination of modifications can beincorporated into the dsRNA. Where multiple modifications are present,they may be the same or different. Modifications to bases, sugarmoieties, the phosphate backbone, and their combinations arecontemplated.

For example, either the 3′ or 5′ terminal regions of the sequences in adsRNA can be phosphorylated or biotinylated. Examples of modificationscontemplated for the phosphate backbone include phosphonates, includingmethylphosphonate, phosphorothioate, and phosphotriester modificationssuch as alkylphosphotriesters, and the like. Examples of modificationscontemplated for the sugar moiety include 2′-alkyl pyrimidine, such as2′-O-methyl, 2′-fluoro, amino, and deoxy modifications and the like.Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Many other modifications are known and can be used so long as the abovecriteria are satisfied

The double-stranded RNA sample can be suitably formulated and introducedinto the environment of the cell by any means that allows for asufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsRNA are known inthe art and can be used so long as dsRNA gains entry to the target cellsso that it can act. For example, dsRNA can be formulated in buffersolutions such as phosphate buffered saline solutions, liposomes,micellar structures, and capsids. Formulations of dsRNA with cationiclipids can be used to facilitate transfection of the dsRNA into cells.Suitable lipids include Oligofectamine, Lipofectamine (LifeTechnologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.),or FuGene 6 (Roche) all of which can be used according to themanufacturer's instructions.

It can be appreciated that the method of introducing dsRNA into theenvironment of the cell will depend on the type of cell and the make upof its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the dsRNA can be added directly to the liquidenvironment of the cells. Lipid formulations can also be administered toanimals such as by intravenous, intramuscular, or intraperitonealinjection, or orally or by inhalation or other methods as are known inthe art. When the formulation is suitable for administration intoanimals such as mammals and more specifically humans, the formulation isalso pharmaceutically acceptable. Pharmaceutically acceptableformulations for administering oligonucleotides are known and can beused. In some instances, it may be preferable to formulate dsRNA in abuffer or saline solution and directly inject the formulated dsRNA intocells, as in studies with oocytes. The direct injection of dsRNAduplexes

Suitable amounts of dsRNA must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual dsRNA species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, more preferredare compositions in which concentrations of about 1 nanomolar or lesscan be used. Even more preferred are methods that utilize aconcentration of about 200 picomolar or less and even a concentration ofabout 50 picomolar or less can be used in many circumstances.

The method can be carried out by addition of the dsRNA compositions toany extracellular matrix in which cells can live provided that the dsRNAcomposition is formulated so that a sufficient amount of the dsRNA canenter the cell to exert its effect. For example, the method is amenablefor use with cells present in a liquid such as a liquid culture or cellgrowth media, in tissue explants, or in whole organisms, includinganimals, such as mammals and especially humans.

As is known, RNAi methods are applicable to a wide variety of genes in awide variety of organisms and the disclosed compositions and methods canbe utilized in each of these contexts. Examples of genes which can betargeted by the disclosed compositions and methods include endogenousgenes which are genes that are native to the cell or to genes that arenot normally native to the cell. Without limitation these genes includeoncogenes, cytokine genes, idiotype (Id) protein genes, prion genes,genes that expresses molecules that induce angiogenesis, genes foradhesion molecules, cell surface receptors, proteins involved inmetastasis, proteases, apoptosis genes, cell cycle control genes, genesthat express EGF and the EGF receptor, multi-drug resistance genes, suchas the MDR1 gene.

Expression of a target gene can be determined by any suitable method nowknown in the art or that is later developed. It can be appreciated thatthe method used to measure the expression of a target gene will dependupon the nature of the target gene. For example, when the target geneencodes a protein the term “expression” can refer to a protein ortranscript derived from the gene. In such instances the expression of atarget gene can be determined by measuring the amount of mRNAcorresponding to the target gene or by measuring the amount of thatprotein. Protein can be measured in protein assays such as by stainingor immunoblotting or, if the protein catalyzes a reaction that can bemeasured, by measuring reaction rates. All such methods are known in theart and can be used. Where the gene product is an RNA species expressioncan be measured by determining the amount of RNA corresponding to thegene product. Several specific methods for detecting gene expression aredescribed in Example 1. The measurements can be made on cells, cellextracts, tissues, tissue extracts or any other suitable sourcematerial.

The determination of whether the expression of a target gene has beenreduced can be by any suitable method that can reliably detect changesin gene expression. Typically, the determination is made by introducinginto the environment of a cell undigested dsRNA such that at least aportion of that dsRNA enters the cytoplasm and then measuring theexpression of the target gene. The same measurement is made on identicaluntreated cells and the results obtained from each measurement arecompared. When the method appears to reduce the expression of the targetgene by about 10% or more (which is equivalent to about 90% or less) ofthe level in an untreated organism, for purposes of this invention, themethod is considered to reduce the expression of the target gene.Typically, the method can be used to reduce the expression of a targetgene by far more than 10%. In some instances the method can be used toreduce the expression by about 50% or more, in more preferred methodsthe expression is reduced by about 75% or more, still more preferableare methods that reduce the expression by about 90% or more, or evenabout 95% or more, or about 99% or more or even by completelyeliminating expression of the target gene.

The dsRNA can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a dsRNA andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a dsRNAeffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

The phrase pharmaceutically acceptable carrier refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA composition may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (see J.Kreuter, (1991) Nanoparticles-preparation and applications. In: M.Donbrow (Ed.) Microcapsules and nanoparticles in medicine and pharmacy.CRC Press, Boca Raton, Fla., pp. 125-14). The polymeric materials whichare formed from monomeric and/or oligomeric precursors in thepolymerization/nanoparticle generation step, are per se known from theprior art, as are the molecular weights and molecular weightdistribution of the polymeric material which a person skilled in thefield of manufacturing nanoparticles may suitably select in accordancewith the usual skill.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration.

In general a suitable dosage unit of dsRNA will be in the range of 0.001to 0.25 milligrams per kilogram body weight of the recipient per day,preferably in the range of 0.01 to 20 micrograms per kilogram bodyweight per day, more preferably in the range of 0.01 to 10 microgramsper kilogram body weight per day, even more preferably in the range of0.10 to 5 micrograms per kilogram body weight per day, and mostpreferably in the range of 0.1 to 2.5 micrograms per kilogram bodyweight per day. Preferably, pharmaceutical composition comprising thedsRNA is administered once daily. However, the therapeutic agent mayalso be dosed in dosage units containing two, three, four, five, six ormore sub-doses administered at appropriate intervals throughout the day.In that case, the dsRNA contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage unit.The dosage unit can also be compounded for a single dose over severaldays, e.g., using a conventional sustained release formulation whichprovides sustained and consistent release of the dsRNA over a severalday period. Sustained release formulations are well known in the art. Inthis embodiment, the dosage unit contains a corresponding multiple ofthe daily dose. Regardless of the formulation, the pharmaceuticalcomposition must contain dsRNA in a quantity sufficient to inhibitexpression of the target gene in the animal or human being treated. Thecomposition can be compounded in such a way that the sum of the multipleunits of dsRNA together contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies, preferably, within a range of circulatingconcentrations that include the ED50 (as determined by known methods)with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound that includes theIC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels of dsRNA in plasma may be measured by standard methods,for example, by high performance liquid chromatography.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the preparation of double-stranded RNAoligonucleotides

Oligonucleotide Synthesis and Purification.

RNA oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie,Methods Mol Biol 1993, 20:81-114; Wincott et al., Nucleic Acids Res1995, 23:2677-84). The oligomers were purified using ion-exchange highperformance liquid chromatography (IE-HPLC) on an Amersham Source 15Qcolumn (1.0 cm×25 cm) (Amersham Pharmacia Biotech, Piscataway, N.J.)using a 15 min step-linear gradient. The gradient varied from 90:10Buffers A:B to 52:48 Buffers A:B, where Buffer A was 100 mM Tris pH 8.5and Buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at260 nm and peaks corresponding to the full-length oligonucleotidespecies were collected, pooled, desalted on NAP-5 columns, andlyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).The CE capillaries had a 100 μm inner diameter and contained ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, ran in an electric field of 444 V/cm anddetected by UV absorbance at 260 nm Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere at least 90% pure as assessed by CE for use in experimentsdescribed below. Compound identity was verified by matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectroscopy on aVoyager DE™ Biospectometry Work Station (Applied Biosystems, FosterCity, Calif.) following the manufacturer's recommended protocol.Relative molecular masses of all oligomers were within 0.2% of expectedmolecular mass.

Preparation of Duplexes. Single-stranded RNA (ssRNA) oligomers wereresuspended at 100 μM concentration in duplex buffer consisting of 100mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense andantisense strands were mixed in equal molar amounts to yield a finalsolution of 50 μM duplex. Samples were heated to 95° C. for 5′ andallowed to cool to room temperature before use. Double-stranded RNA(dsRNA) oligomers were stored at −20° C. Single-stranded RNA oligomerswere stored lyophilized or in nuclease-free water at −80° C.

Nomenclature.

For consistency, the following nomenclature has been employed throughoutthe Examples. Names given to duplexes indicate the length of theoligomers and the presence or absence of overhangs. A “21+2” duplexcontains two RNA strands both of which are 21 nucleotides in length,also termed a 21-mer siRNA duplex, and having a 2 base 3′-overhang. A“21-2” design is a 21-mer siRNA duplex with a 2 base 5′-overhang. A 21-0design is a 21-mer siRNA duplex with no overhangs (blunt). A “21+2UU” isa 21-mer duplex with 2-base 3′-overhang and the terminal 2 bases at the3′-ends are both U residues (which may result in mismatch with targetsequence).

Example 2

This example demonstrates that dsRNAs having strands that are 25nucleotides in length or longer have surprisingly increased potency inmammalian systems than known 21-23-mer siRNAs.

Cell Culture, Transfection, and EGFP Assays.

Human embryonic kidney (HEK) 293 cells were grown in DMEM mediumsupplemented with 10% fetal bovine serum (FBS) (Irvine Scientific, SantaAna, Calif.). Transfections were done at 90% confluence in 24-wellplates using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instructions. Briefly, 50 μl of Opti-MEM media wasmixed with nucleic acids, including siRNA duplexes and/or 100-200 ngplasmid pEGFP-C1 (Clontech, Palo Alto, Calif.) for 5 min. Nucleic acidswere then mixed with 50 μl of Opti-MEM media that had been pre-mixedwith 1.5 μl of Lipofectamine 2000 and incubated at room temperature for15 min. The lipid-nucleic acid mixtures were added to cells afterremoval of old media and swirled and then an additional 0.4 ml of mediapre-warmed to 37° C. was added. Incubation was continued at 37° C. andcells were assayed for fluorescence at the times indicated. Each assaywas performed in triplicate. EGFP expression levels were measured bydirect fluorescence in a fluorescence-activated cell sorter (FACS)(Moslo-MLS, Dako Cytomation, Fort Collins, Colo.) in the City of HopeCytometrics Core Facility (Duarte, Calif.). EGFP expression was measuredas the percentage of cells showing detectable fluorescence abovebackground (mock-transfected negative control cells).

NIH 3T3 cells that stably expressed EGFP (Kim and Rossi, 2003, AntisenseNucleic Acid Drug Dev., 13:151-155) were grown in DMEM mediasupplemented with 10% FBS. Cells were plated at 30% density on 24-wellplates and transfected with siRNA alone without reporter plasmid usingthe same method described above. Media was changed at 24 h and EGFPassays were performed at 3, 6 and 9 days post-transfection. At 3 dayspost-transfection, 1×10⁵ cells were used for extract preparation and1×10⁴ cells were re-plated and continued incubation for later assay. Atday 6, 1×10⁵ cells were used for extract preparation, and 1×10⁴ cellswere re-plated and continued incubation for later assay. At day 9, 1×10⁵cells were used for extract preparation. For extract preparation, 1×10⁵cells were suspended in 300 μl phosphate buffered saline (PBS) andsonicated for 10 sec. Cells were centrifuged at 14,000 g for 2 min andcell supernatant was recovered for fluorometry. EGFP fluorescence wasexamined using a VersaFluor Cuvette Fluorometer (Bio-Rad, Hercules,Calif.) using excitation filter D490 and emission filter D520.Percentage of EGFP expression was determined relative to extractprepared from non-transfected control cells.

In addition, cells were directly examined by fluorescence microscopyusing a Nikon Eclipse TE2000-S(Nikon Instech Co., Kanagawa, JP) usingthe program Spot v3.5.8. Images were digitally captured with identicalexposure times so that comparisons between cells samples could be made.

Nucleic Acid Reagents.

The reporter system employed EGFP either as a transfection plasmidvector pEGFP-C1 (Clontech, Palo Alto, Calif.) or as a stabletransformant in an NIH 3T3 cell line. The coding sequence of EGFP isshown below, from Genbank accession #U55763. The ATG start codon and TAAstop codons are highlighted in bold font and sites target by siRNAreagents are underscored.

SEQ ID NO. 1: atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggca tggacgagctgtacaagtaa

Site-1 used for siRNA targeting in EGFP was:

SITE 1:  (SEQ ID NO: 67) 5′ GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3′

Site-2 used for siRNA targeting in EGFP was:

SITE 2:  (SEQ ID NO: 68) 5′ UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG 3′

RNA duplexes were synthesized and prepared as described in Example 1.RNA duplexes targeting EGFP Site-1 are summarized in Table 1 below. Somesequences had the dinucleotide sequence “UU” placed at the 3′-end of thesense strand (Elbashir et al., 2001, EMBO J., 20:6877-6888; Hohjoh,2002, FEBS Lett., 521:195-199). Mismatches that resulted from including3′-terminal “UU” or where a mismatch was intentionally positioned arehighlighted in bold and underscored.

TABLE 1 Summary of Oligonucleotide Reagents, EGFP Site-1 Sequence NameSEO ID NOo. 5′ GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3′ EGFP Site-1SEQ ID NO: 67 5′ GCAAGCUGACCCUGAAGUUCA EGFPS1-21 - 2 SEQ ID No. 2 3′UUCGACUGGGACUUCAAGUAG SEQ ID NO. 3 5′ AAGCUGACCCUGAAGUUCAUC EGFPS1-21 +0 SEQ ID No. 4 3′ UUCGACUGGGACUUCAAGUAG SEQ ID NO. 5 5′GCUGACCCUGAAGUUCAUCUG EGFPS1-21 + 2(7) SEQ ID No. 6 3′UUCGACUGGGACUUCAAGUAG SEQ ID NO. 7 5′ GCAAGCUGACCCUGAAGUUCAU UEGFPS1-23 - 2UU SEQ ID No. 8 3′ UUCGACUGGGACUUCAAGUAGAC SEQ ID NO. 9 5′GCUGACCCUGAAGUUCAUCUGUU EGFPS1-23 + 2UU SEQ ID No. 10 3′UUCGACUGGGACUUCAAGUAGAC SEQ ID NO. 11 5′ GCAAGCUGACCCUGAAGUUCAU U UEGFPS1-24 - 2UU SEQ ID No. 12 3′ UUCGACUGGGACUUCAAGUAGACG SEQ ID NO. 135′ GCUGACCCUGAAGUUCAUCUGCUU EGFPS1-24 + 2UU SEQ ID No. 14 3′UUCGACUGGGACUUCAAGUAGACG SEQ ID NO. 15 5′ GCAAGCUGACCCUGAAGUUCAUCU UEGFPS1-25 - 2UU SEQ ID No. 16 3′ UUCGACUGGGACUUCAAGUAGACGU SEQ ID NO. 175′ GCUGACCCUGAAGUUCAUCUGCAUU EGFPS1-25 + 2UU SEQ ID No. 18 3′UUCGACUGGGACUUCAAGUAGACGU SEQ ID NO. 19 5′ AAGCUGACCCUGAAGUUCAUCUGCACEGFPS1-26 + 0 SEQ ID No. 20 3′ UUCGACUGGGACUUCAAGUAGACGUG SEQ ID NO. 215′ AAGCUGACCCUGAAGUUCAUCUGC UU EGFPS1-26 + 0UU SEQ ID No. 22 3′UUCGACUGGGACUUCAAGUAGACGUG SEQ ID NO. 23 5′ GCAAGCUGACCCUGAAGUUCAUCU UUEGFPS1-26 - 2UU SEQ ID No. 24 3′ UUCGACUGGGACUUCAAGUAGACGUGSEQ ID NO. 25 5′ GCUGACCCUGAAGUUCAUCUGCACUU EGFPS1-26 + 2UUSEQ ID No. 26 3′ UUCGACUGGGACUUCAAGUAGACGUG SEQ ID NO. 27 5′AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0 SEQ ID No. 28 3′UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID NO. 29 5′ AAGCUGACCCUGAAGUUCAUCUGCAUU EGFPS1-27 + 0UU SEQ ID No. 30 3′ UUCGACUGGGACUUCAAGUAGACGUGGSEQ ID NO. 31 5′ GCAAGCUGACCCUGAAGUUCAUCUG UU EGFPS1-27 - 200SEQ ID No. 32 3′ UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID NO. 33 5′GCUGACCCUGAAGUUCAUCUGCAC A UU EGFPS1-27 + 2UU, SEQ ID No. 34 3′UUCGACUGGGACUUCAAGUAGACGUGG mut SEQ ID NO. 35 5′AAGCUGACCCUGUUCAUCAUCUGCACC EGFPS1-27 + 0-mut SEQ ID No. 36 3′UUCGACUGGGACAAGUAGUAGACGUGG SEQ ID NO. 37 5′AAGCUGACCCUGAAGUUCAUCUGCACCA EGFPS1-28 + 0 SEQ ID No. 38 3′UUCGACUGGGACUUCAAGUAGACGUGGU SEQ ID NO. 39 5′AAGCUGACCCUGAAGUUCAUCUGCACCAC EGFPS1-29 + 0 SEQ ID No. 40 3′UUCGACUGGGACUUCAAGUAGACGUGGUG SEQ ID NO. 41 5′AAGCUGACCCUGAAGUUCAUCUGCACCACC EGFPS1-30 + 0 SEQ ID No. 42 3′UUCGACUGGGACUUCAAGUAGACGUGGUGG SEQ ID NO. 43

Results.

HEK 293 cells were mock transfected (negative control), transfected with200 ng EGFP reporter plasmid alone (positive control), or reporterplasmid+siRNA duplexes at varying concentrations. EGFP expression wasassessed using the FACS assay at 24 h post-transfection. Results areshown in FIG. 1. At 50 nM concentration (FIG. 1A), a 21-mer siRNA duplexwith 2-base 3′-overhang (21+2 design) (SEQ ID NoS. 6/7) showed about a70% reduction in EGFP expression while longer duplexes were more potent.25-mer siRNA duplexes (SEQ ID Nos. 16/17 and 18/19) and longer (SEQ IDNos. 20/21, 24/25, 26/27, 30/31, 32/33, and 34/35) suppressed EGFP belowdetection limits Typically, 21-mer siRNA oligos are employed at 10-100nM concentration by those skilled in the art. At 1 nM concentration(FIG. 1B), a concentration much lower than is typically employed today,the 21-mer duplex showed only about a 40% reduction in EGFP expressionwhile longer duplexes continued to suppress EGFP below detection limits.At 200 pM concentration (FIG. 1C), the 21-mer duplex had no effect onEGFP expression while the blunt 27-mer duplex continued to suppress EGFPbelow detection limits. At 50 pM concentration (FIG. 1D), the 27-merblunt duplex (SEQ ID No. 30/31) suppressed EGFP expression by about 90%or more. The longer and shorter duplexes tested (26-mers SEQ ID No.22/23, 24/25, 26/27; 29-mer SEQ ID No. 40/41; or 30-mer SEQ ID No.42/43) were slightly less effective than the blunt 27-mer.

This experiment was repeated using NIH 3T3 cells that stably expressEGFP. EGFP protein was detected in cellular extracts using a cuvettefluorometer as described above. The dose response curves for 21-merduplex with 2-base 3′-overhang (SEQ ID No. 6/7), 25-mer duplex with2-base 5′-overhang (SEQ ID Nos. 16/17), and blunt 27-mer duplex (SEQ IDNos. 30/31) are shown in FIG. 2. The exact level of suppression variedbetween experiments done using stably transfected 3T3 cells comparedwith transiently transfected HEK 293 cells (FIG. 1), however qualitativetrends were identical.

This example demonstrates that the longer dsRNAs of the invention haveabout a 100-fold or more higher potency than traditional 21-mer siRNAs.The enhanced effect was first seen at about a 25-mer length and maximalpotency was achieved with a 27-mer. Potent RNAi effects were observedfor 30-mer duplexes (the longest compounds tested herein), with noapparent toxicity to either the HEK 293 cells or NIH 3T3 cells.Furthermore, as duplex length was increased above 25-mer length(presumably when the duplex is sufficiently long to be a Dicersubstrate), a 2-base 3′-overhang (as taught in the prior art) is nolonger necessary. In the present experiments 25-mer duplexes with a2-base 5′-overhang had similar potency as did blunt ended duplexes orduplexes with a 2-base 3′-overhang. In the current experimental system,the 27-mer blunt duplex showed greatest potency.

Within the set of 27-mer RNA duplexes tested in this example, duplexesthat included base mismatches between the sense and antisense strands(SEQ ID Nos. 30/31, 32/33, 34/35) were more potent than the duplexhaving perfect complementarity (SEQ ID Nos. 28/29). These duplexes(27+0UU, 27+2UU, and 27-2UU) had 1 or 2 mismatches at the 3′-end of thesense strand. The set of 27-mer duplexes were compared for effectivesuppression of EGFP expression in the HEK 293 cell transienttransfection assay and the results are shown in FIG. 3. Duplex 27+0UU(SEQ ID Nos. 30/31) was most potent.

The use of mismatches or decreased thermodynamic stability (specificallyat the 3′-sense/5′-antisense position) has been proposed to facilitateor favor entry of the antisense strand into RISC (Schwarz et al., 2003,Cell, 115:199-208; Khvorova et al., 2003, Cell, 115:209-216), presumablyby affecting some rate-limiting unwinding steps that occur with entry ofthe siRNA into RISC. Because of this terminal base composition has beenincluded in design algorithms for selecting active 21-mer siRNA duplexes(Ui-Tei et al., 2004, Nucleic Acids Res., 32:936-948; Reynolds et al.,2004, Nat. Biotechnol., 22:326-330). It has been proposed that the27-mer duplexes employed in this example do not directly enter RISC butfirst are cleaved by Dicer into 21-mer siRNAs. With Dicer cleavage, thesmall end-terminal sequence which contains the mismatches will either beleft unpaired with the antisense strand (become part of a 3′-overhang)or be cleaved entirely off the final 21-mer siRNA. These “mismatches”,therefore, do not persist as mismatches in the final RNA component ofRISC. It was surprising to find that base mismatches or destabilizationof segments at the 3′-end of the sense strand of a Dicer substrateimprove the potency of synthetic duplexes in RNAi, presumably byfacilitating processing by Dicer.

Example 3

This example demonstrates that the use of 25-30 nucleotide RNA duplexesallows gene targeting at a site that could not be effectively targetedusing traditional siRNA 21-mer designs.

It is currently expected in the art that the majority of 21-mer siRNAduplexes targeted to sites within a given target gene sequence will beineffective (Holen et al., 2002, Nucleic Acids Res., 30:1757-1766).Consequently, a variety of sites are commonly tested in parallel orpools containing several distinct siRNA duplexes specific to the sametarget with the hope that one of the reagents will be effective (Ji etal., 2003, FEBS Lett., 552:247-252). To overcome the need to pool orengage in large scale empiric testing, complex design rules andalgorithms have been devised to increase the likelihood of obtainingactive RNAi effector molecules (Schwarz et al., 2003, Cell, 115:199-208;Khvorova et al., 2003, Cell, 115:209-216; Ui-Tei et al., 2004, NucleicAcids Res., 32:936-948; Reynolds et al., 2004, Nat. Biotechnol.,22:326-330). These design rules significantly limit the number of sitesamenable to RNAi knockdown within a given target gene. In fact, thedesign can be overly restrictive in situations demanding the suppressionof specific alleles or isoforms. Moreover, the rules are not perfect anddo not always provide active siRNA effector molecules. This exampleshows that the use of dsRNA duplexes of the present invention allow RNAitargeting at sites that were ineffectively targeted by previously known21-mer siRNA reagents. This result minimizes the need for empiricallytesting multiple sites or using pooled reagent sets.

Nucleic Acid Reagents. The reporter system employed EGFP as in SEQ IDNo. 1 above. Site-2 in EGFP, as shown in Example 1, was targeted. RNAduplexes were synthesized and prepared as described in Example 1. RNAduplexes targeting EGFP Site-2 are summarized in Table 2 below. DuplexEGFPS2-27+0 mm was a blunt 27-mer duplex with a 2 base mismatch at theterminal 2 bases of the sense strand. These bases are shown in bold andunderscored.

TABLE 2 Summary of Oligonucleotide Reagents, EGFP Site-2 Sequence NameSEQ ID No. 5′ UGAAGCAGCACGACUUCUUCAAGUCCGCCAUG 3′ EGFP Site-2SEQ ID NO: 68 5′ GCAGCACGACUUCUUCAAGUU EGFPS2-21 + 2 SEQ ID No. 44 3′UUCGUCGUGCUGAAGAAGUUC SEQ ID No. 45 5′ AAGCAGCACGACUUCUUCAAGUCCGCCEGFPS2-27 + 0 SEQ ID No. 46 3′ UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID No. 475′ AAGCAGCACGACUUCUUCAAGUCCG GG EGFPS2-27 + 0mm SEQ ID No. 48 3′UUCGUCGUGCUGAAGAAGUUCAGGCGG SEQ ID No. 49

Results.

HEK 293 cells were mock transfected (negative control), transfected with200 ng EGFP reporter plasmid alone (positive control), or reporterplasmid+RNA duplexes at varying concentrations as described previously.EGFP expression was assessed using the FACS assay at 24 hpost-transfection. Results are shown in FIG. 4. At 10 nM concentration,the traditional 21-mer siRNA duplex with 2-base 3′-overhangs targeted toSite-2 in the EGFP gene (SEQ ID Nos. 44/45) did not detectably reduceEGFP expression. In contrast, a 10 nM concentration of the longer 27-merduplex RNA (SEQ ID No. 46/47) reduced EGFP by about 80% or more and 10nM of a related 27-mer (SEQ ID No. 48/49) reduced EGFP by about 90% ormore. As in Example 2 above, 3′- or 5′-overhangs did not improveactivity over the blunt ended version. The 27-mer with the 2-basemismatch in the sense strand (SEQ ID Nos. 48/49) showed improvedactivity as compared to the perfectly matched 27-mer (SEQ ID Nos.46/47). It is possible that destabilization of the RNA duplex at thisposition improves efficiency of cleavage by Dicer.

This example demonstrates that dsRNAs of the invention can efficientlytarget sites within the EGFP gene that were previously considered poortargets by previously known methods. Use of the method of the inventionwill therefore simplify site selection and design criteria for RNAi.This example also shows that the intentional placement of mismatches atthe 3′-terminus of the sense strand increases the potency of the 27-merduplex.

Example 4

This example demonstrates the use of the disclosed dsRNA duplexes toreduce expression of the human HNRPH1 gene in HEK 293 cells.

Western Blot.

HEK 293 cells were cultured in a 6-well plate. At 30% confluence, cellswere transfected with iRNA duplexes as outlined in Example 2 aboveexcept that all reagents were used at 5-fold higher volume due to thelarger scale of the cultures. Cells were harvested at 72 h in 300 μlphosphate buffered saline (PBS) and sonicated for 10 sec. Cell lysateswas centrifuged for 2 min at 14,000 g and the supernatant was collected.Aliquots of 2 μl were taken from the cleared lysates which were run on a10% SDS-PAGE gel. The HNRPH1 gene product was detected using a rabbitpolyclonal anti-HNRPH1 antiserum and an anti-rabbit antibody conjugatedwith alkaline phosphatase (Sigma, St. Louis, Mo.). As control, β-actinwas detected by a murine anti-human actin antibody (Sigma, St. Louis,Mo.) and anti-mouse antibody conjugated with alkaline phosphatase(Sigma, St. Louis, Mo.), as previously described (Markovtsov et al.,2000, Mol. Cell Biol., 20:7463-79).

Nucleic Acid Reagents.

The coding sequence of Homo sapiens heterogeneous nuclearribonucleoprotein H1 (HNRPH1) mRNA is shown (Genbank accession No.NM_005520) below. The ATG start codon and TAA stop codons arehighlighted in bold font and site target by siRNA reagents isunderscored.

SEQ ID No. 50: ttttttttttcgtottagccacgcagaagtcgcgtgtctagtttgtttcgacgccggaccgcgtaagagacgatgatgttgggcacggaaggtggagagggattcgtggtgaaggtccggggcttgccctggtcttgctcggccgatgaagtgcagaggtttttttctgactgcaaaattcaaaatggggctcaaggtattcgtttcatctacaccagagaaggcagaccaagtggcgaggcttttgttgaacttgaatcagaagatgaagtcaaattggccctgaaaaaagacagagaaactatgggacacagatatgttgaagtattcaagtcaaacaacgttgaaatggattgggtgttgaagcatactggtccaaatagtcctgacacggccaatgatggctttgtacggcttagaggacttccctttggatgtagcaaggaagaaattgttcagttcttctcagggttggaaatcgtgccaaatgggataacattgccggtggacttccaggggaggagtacgggggaggccttcgtgcagtttgcttcacaggaaatagctgaaaaggctctaaagaaacacaaggaaagaatagggcacaggtatattgaaatctttaagagcagtagagctgaagttagaactcattatgatccaccacgaaagcttatggccatgcagcggccaggtccttatgacagacctggggctggtagagggtataacagcattggcagaggagctggctttgagaggatgaggcgtggtgcttatggtggaggctatggaggctatgatgattacaatggctataatgatggctatggatttgggtcagatagatttggaagagacctcaattactgtttttcaggaatgtctgatcacagatacggggatggtggctctactttccagagcacaacaggacactgtgtacacatgcggggattaccttacagagctactgagaatgacatttataattttttttcaccgctcaaccctgtgagagtacacattgaaattggtcctgatggcagagtaactggtgaagcagatgtcgagttcgcaactcatgaagatgctgtggcagctatgtcaaaagacaaagcaaatatgcaacacagatatgtagaactcttcttgaattctacagcaggagcaagcggtggtgcttacgaacacagatatgtagaactcttcttgaattctacagcaggagcaagcggtggtgcttatggtagccaaatgatgggaggcatgggcttgtcaaaccagtccagctacgggggcccagccagccagcagctgagtgggggttacggaggcggctacggtggccagagcagcatgagtggatacgaccaagttttacaggaaaactccagtgattttcaatcaaacattgcataggtaaccaaggagcagtgaacagcagctactacagtagtggaagccgtgcatctatgggcgtgaacggaatgggagggttgtctagcatgtccagtatgagtggtggatggggaatgtaattgatcgatcctgatcactgactcttggtcaacctttttttttttttttttttctttaagaaaacttcagtttaacagtttctgcaatacaagcttgtgatttatgcttactctaagtggaaatcaggattgttatgaagacttaaggcccagtatttttgaatacaatactcatctaggatgtaacagtgaagctgagtaaactataactgttaaacttaagttccagcttttctcaagttagttataggatgtacttaagcagtaagcgtatttaggtaaaagcagttgaattatgttaaatgttgccctttgccacgttaaattgaacactgttttggatgcatgttgaaagacatgcttttattttttttgtaaaacaatataggagctgtgtctactattaaaagtgaaacattttggcatgtttgttaattctagtttcatttaataacctgtaaggcacgtaagtttaagctttttttttttttaagttaatgggaaaaatttgagacgcaataccaatacttaggattttggtcttggtgtttgtatgaaattctgaggccttgatttaaatctttcattgtattgtgatttccttttaggtatattgcgctaagtgaaacttgtcaaataaatcctccttttaaaaactg c

RNA duplexes were synthesized and prepared as described in Example 1.RNA duplexes targeting HNRPH1 are summarized in Table 3 below.

TABLE 3 Summary of Oligonucleotide Reagents, HNRPH1 Site-1 Sequence NameSEQ ID No. 5′ UGAACUUGAAUCAGAAGAUGAAGUCAAAUUGGC 3′ HNRPH1 Site-1SEQ ID NO: 69 5′ CUUGAAUCAGAAGAUGAAGUU HNRPH1-21 + 2 SEQ ID No. 51 3′UUGAACUUAGUCUUCUACUUC SEQ ID No. 52 5′ AACUUGAAUCAGAAGAUGAAGUCAAAUHNRPH1-27 + 0 SEQ ID No. 53 3′ UUGAACUUAGUCUUCUACUUCAGUUUA SEQ ID No. 54

HEK 293 cells were transfected with EGFP-specific siRNA (SEQ ID No. 6/7)(negative control) and an HNRPH1 specific 21-mer siRNA duplex (SEQ IDNos. 51/52) at varying concentrations, or with an HNRPH1 specific 27-mersiRNA duplex (SEQ ID Nos. 53/54) at varying concentrations, as describedpreviously. HNRPH1 expression was assessed by Western Blot assay at 72 hpost-transfection. Results are shown in FIG. 5. As shown, only a slightdecrease in HNRPH1 protein levels occurred after treatment with 20 nM ofthe 21-mer siRNA (SEQ ID Nos 51/52) while significant inhibition wasseen using 1 nM of the 27-mer dsRNA (SEQ ID Nos 53/54) and almostcomplete elimination of HNRPH1 protein was achieved using 5 nM of the27-mer RNA duplex Improved reduction in gene expression by RNAi methodsis therefore also seen for human genes using the method of theinvention.

Example 5

This example demonstrates a method for determining whether a dsRNAserves as s substrate for Dicer.

In Vitro Dicer Assay.

Recombinant human Dicer enzyme (Gene Therapy Systems, San Diego, Calif.)was incubated with synthetic duplex RNA oligonucleotides according tothe manufacturer's instructions. Briefly, 2 units of Dicer was incubatedin a buffer supplied by the manufacturer with 250 pmoles of RNA duplexin a 50 μl volume (5 μM RNA concentration) for 18 h at 37° C. Half ofeach reaction was separated on non-denaturing PAGE (10% acrylamide) andvisualized using ethidium bromide staining with UV excitation.

Results.

RNA duplexes tested included 21-mer (SEQ ID No. 6/7), 25-mer (SEQ ID No.18/19), 26-mer (SEQ ID No. 24/25), 27-mer (SEQ ID No. 30/31), and 29-mer(SEQ ID No. 40/41). The duplexes were subjected to Dicer digestion invitro and visualized by PAGE. Results are shown in FIG. 6. As shown, the21-mer RNA duplex did not react with Dicer. The 25-mer, 26-mer, 27-mer,and 29-mer duplexes all reacted with Dicer and were digested to a 21-mersize product, predominantly.

This example shows that the longer RNA duplexes used in the method ofthe invention are substrates for the Dicer endoribonuclease.

Example 6

This example demonstrates that 27-mer duplexes have more RNAi activitythan any of the shorter 21-mer duplexes that they encompass.

Theoretically, a variety of short 21-mer siRNAs could result from theaction of Dicer on longer duplex RNAs. For example, based upon theantisense strand, 7 different 21-mer species could result fromdegradation of a 27-mer sequence. It is possible that one of these21-mers (or a combination of 21-mers) accounts for the activity observedwith the previously tested 27-mer dsRNA. This example shows that nosingle 21-mer duplex or mixture of 21-mers resulting from degradation ofa 27-mer sequence functions as effectively as its parent 27-mer duplexat reducing EGFP expression.

Nucleic Acid Reagents.

RNA duplexes were prepared as described in Example 1. The sequences of aset of 21-mer RNA duplexes from within EGFP Site-1 were prepared. Theduplexes are listed below in Table 4. The 21-mer duplexes are alignedbeneath the parent 27-mer to illustrate their relative positioning. The27-mer blunt duplex (SEQ ID No. 28/29) and the 21-mer duplex 21+2(7)(SEQ ID No. 6/7) are shown in Example 2 (Table 1) and were also used.

TABLE 4 Summary of Oligonucleotide Reagents, EGFP Site-I, Tiled SetSequence Name SEQ ID No. 5′ GCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGC 3′EGFP Site-1 SEQ ID NO: 67 5′ AAGCUGACCCUGAAGUUCAUCUGCACC EGFPS1-27 + 0SEQ ID No. 28 3′ UUCGACUGGGACUUCAAGUAGACGUGG SEQ ID No. 29 5′CCUGAAGUUCAUCUGCACCAC EGFPS1-21 + 2(1) SEQ ID No. 55 3′UGGGACUUCAAGUAGACGUGG SEQ ID No. 56 5′ CCCUGAAGUUCAUCUGCACCA EGFPS1-21 +2(2) SEQ ID No. 57 3′ CUGGGACUUCAAGUAGACGUG SEQ ID No. 58 5′ACCCUGAAGUUCAUCUGCACC EGFPS1-21 + 2(3) SEQ ID No. 59 3′ACUGGGACUUCAAGUAGACGU SEQ ID No. 60 5′ GACCCUGAAGUUCAUCUGCAC EGFPS1-21 +2(4) SEQ ID No. 61 3′ GACUGGGACUUCAAGUAGACG SEQ ID No. 62 5′UGACCCUGAAGUUCAUCUGCA EGFPS1-21 + 2(5) SEQ ID No. 63 3′CGACUGGGACUUCAAGUAGAC SEQ ID No. 64 5′ CUGACCCUGAAGUUCAUCUGC EGFPS1-21 +2(6) SEQ ID No. 65 3′ UCGACUGGGACUUCAAGUAGA SEQ ID No. 66 5′GCUGACCCUGAAGUUCAUCUG EGFPS1-21 + 2(7) SEQ ID No. 6 3′UUCGACUGGGACUUCAAGUAG SEQ ID No. 7

The each of the 21-mer duplexes from Table 4 was transfectedindividually or together as a pool into HEK 293 cells with 200 ng ofEGFP reporter plasmid as described previously. The result from eachtransfection was compared with the 27-mer duplex (SEQ ID No. 28/29). Therelative EGFP expression from each experiment is shown in FIG. 7. Atconcentrations of 50 or 200 pM, none of the individual 21-mer duplexesor the pooled set of 7 21-mer duplexes showed activity comparable withthe 27-mer duplex. For pools, 50 pM and 200 pM represent the totalconcentration of all RNAs transfected together, rather than forindividual duplexes. FIG. 7 shows that the potency of the 27-mer duplexwas much higher than for any of the shorter 21-mer sequences, whichincluded every possible 21-mer duplex that could result from degradationof the parent 27-mer.

The activity of “diced” products made from digestion of the 27-merduplex with recombinant Dicer enzyme in vitro was compared with theparent 27-mer compound in RNAi assays. The 27-mer duplex (SEQ ID No.28/29) was degraded using Dicer as described in Example 5 above andfragments (“diced” products) were diluted and directly used intransfection experiments. EGFP expression levels were measured followingtransfection of HEK 293 cells with 200 ng EGFP reporter plasmid with a21-mer duplex (SEQ ID No. 6/7), a 27-mer duplex (SEQ ID No. 28/29),products of in vitro Dicer degradation (“diced” products), a mutant27-mer with 4 base central mismatch (SEQ ID No. 36/37), and the pooledset of 7 21-mer duplexes (SEQ ID Nos. 6/7 and 55/56, 57/58, 59/60,61/62, 63/64, 65/66). Results are shown in FIG. 8. Again, the 27-merduplex was the most potent reagent in reducing EGFP expression. The“diced” products were more effective than the set of pooled 21-merduplexes. One explanation for this result is that the in vitro dicingreaction was incomplete and some intact 27-mer remains even after 18 hincubation (residual 27-mer is seen in FIG. 6).

This example provides another demonstration that the improved potency ofdsRNA 27-mers is not derived from a highly active individual or a pooledset of short 21-mer duplexes.

Example 7

This example demonstrates that gene suppression using the 27-merduplexes of the invention last twice as long as suppression achievedusing 21-mer duplexes.

Suppression of gene expression using synthetic siRNA typically has aduration of 3-4 days in tissue culture (Chiu and Rana, 2002, Mol. Cell,10:549-561). Methods that increase the duration of the RNAi effect wouldimprove the functional utility of RNAi as an experimental tool in tissueculture and would be beneficial for use of RNAi in vivo.

NIH 3T3 cells were transfected with 5 nM of either the 21-mer duplex(SEQ ID No. 6/7) or the 27-mer duplex (SEQ ID No. 30/31). Cell extractswere prepared and measured for EGFP protein expression in a cuvettefluorometer (as described in Example 2 above) at 2, 4, 6, 8, and 10 dayspost-transfection. Results are shown in FIG. 9. In addition, images ofcells that were transfected in parallel using 1 nM siRNAs were obtainedusing fluorescence microscopy (as described in Example 2) and the imagesare shown in FIG. 10. EGFP expression was suppressed to about 70% ofcontrol levels at day 4 but returned to about 80% of control levels atday 6 and was at control levels at day 8. In contrast, suppression usingthe 27-mer duplex was about 90% or more at day 8 and was still at about70% on day 10.

The 27-mer duplexes used in the present example demonstrates suppressionof gene expression for at least twice the duration seen using 21-merduplexes.

Example 8

This example demonstrates that the dsRNA duplexes of the invention donot activate the interferon response.

Historically, long double stranded RNA was considered to be ineffectiveas an agent for reducing gene expression in mammalian cells because ittends to activate interferon pathway responses and lead to a variety ofmetabolic disturbances in cells which are not sequence specific. Short21-mer siRNAs were considered useful for RNA I experiments because, inaddition to suppressing gene expression, they avoid interferonactivation. Because the more active double stranded RNAs of thisinvention are longer than known siRNA duplexes, they were examinedfurther to show that they do not activate interferon. Duplexes of up toabout 30-mer lengths were tested.

Interferon and PKR Assays.

HEK 293 cells were transfected with 20 nM T7 ssRNA (Kim et al., 2004,Nat. Biotechnol., 22:321-325), 21-mer RNA duplex (SEQ ID No. 6/7), or27-mer RNA duplex (SEQ ID No. 30/31) as described in Example 2 above.Culture medium was collected at 24 h and subjected to interferon alphaand beta ELISA assays (Research Diagnostics, Inc., Flanders, N.J.)according to the manufacturer's instructions, as previously described(Kim et al., 2004, Nat. Biotechnol., 22:321-325).

Human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) wasassayed using the PKR activation assay (Gunnery et al., 1998, Methods,15:189-98) in HEK 293 cell extracts. HEK 293 cells were transfected asdescribed in Example 2 with 20 nM of each RNA and extracts were prepared18 h post-transfection.

Results.

HEK 293 cells were transfected with ssRNA, 21-mer duplex, 27-mer duplex,or no RNA (negative control, mock transfection) as described above. Asshown in FIG. 11, high levels of interferon alpha (FIG. 11A) andinterferon beta (FIG. 11B) were detected after transfection with ssRNAhowever no interferon was detected when 21-mer or 27-mer RNAs weretransfected.

HEK 293 cells were transfected with 400 bp EGFP dsRNA (at 20 nM), or 20nM of chemically synthesized short RNA duplexes including 21+2 (SEQ IDNo. 6/7), 25+2 (SEQ ID No. 18/19), 25-2 (SEQ ID No. 16/17), 27+2 (SEQ IDNo. 34/35), and 27-2 (SEQ ID No. 32/33). Results of the PKR activationassay are shown in FIG. 12. The long dsRNA resulted in strong PKRactivation (positive control) while all of the short synthetic RNAsshowed no evidence for PKR activation.

We conclude that the longer synthetic RNAs used in the invention forimproved RNAi mediated suppression of gene expression do not activateinterferon responses and therefore should be usable in a wide variety ofmammalian systems.

Example 9

This example demonstrates a method for determining an effective dose ofthe dsRNA of the invention in a mammal. A therapeutically effectiveamount of a composition containing a sequence that encodes a dsRNA,(i.e., an effective dosage), is an amount that inhibits expression ofthe product of the target gene by at least 10 percent. Higherpercentages of inhibition, e.g., 20, 50, 90 95, 99 percent or higher maybe preferred in certain circumstances. Exemplary doses include milligramor microgram amounts of the molecule per kilogram of subject or sampleweight (e.g., about 1 microgram per kilogram to about 5 milligrams perkilogram, about 100 micrograms per kilogram to about 0.5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The compositions can be administered one or more times perweek for between about 1 to 10 weeks, e.g., between 2 to 8 weeks, orbetween about 3 to 7 weeks, or for about 4, 5, or 6 weeks, as deemednecessary by the attending physician. Treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments.

Appropriate doses of a particular dsRNA composition depend upon thepotency of the molecule with respect to the expression or activity to bemodulated. One or more of these molecules can be administered to ananimal, particularly a mammal, and especially humans, to modulateexpression or activity of one or more target genes. A physician may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of other factors includingthe severity of the disease, previous treatment regimen, other diseasespresent, off-target effects of the active agent, age, body weight,general health, gender, and diet of the patient, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The efficacy of treatment can be monitored by measuring the amount ofthe target gene mRNA (e.g. using real time PCR) or the amount of productencoded by the target gene, such as by Western blot analysis. Inaddition, the attending physician can monitor the symptoms associatedwith the disease or disorder afflicting the patient and compare withthose symptoms recorded prior to the initiation of treatment.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

-   Amarzguioui et al., Tolerance for Mutation and Chemical    Modifications in a siRNA, Nucleic Acids Research, 2003, Vol. 31, No.    2, 589-595.-   Bernstein, E., Caudy, A. A., Hammond, S. M., and    Hannon, G. J. (2001) Role for a bidentate ribonuclease in the    initiation step of RNA interference. Nature, 409:363-366.-   Braasch et al., RNA Interference in Mammalian Cells by    Chemically-Modified RNA, Biochemistry, 2003, 42, 7967-7975.-   Bridge et al., Induction of an Interferon Response by RNAi Vectors    in Mammalian Cells, Nature Genetics, 2003, Vol. 34, No. 3, 263-264.-   Caplen, N. J., Parrish, S., Imani, F, Fire, A., and    Morgan, R. A. (2001) Specific inhibition of gene expression by small    double-stranded RNAs in invertebrate and vertebrate systems. Proc.    Natl. Acad. Sci. USA, 98:9742-9747.-   Chang et al., Gene Expression from Both Intronless and    Intron-Containing Rous Sarcoma Virus Clones is Specifically    Inhibited by Anti-Sense RNA, Molecular and Cellular Biology, 1985,    Vol. 5, No. 9, 2341-2348.-   Check, RNA to the Rescue?, Nature, 2003, 425, 10-12.-   CHIU et al., siRNA Function in RNAi: A Chemical Modification    Analysis, R N A, 2003, 9:1034-1048.-   Chiu, Y. L., and Rana, T. R. (2002) RNAi in human cells: basic    structural and functional features of small interfering RNA. Mol.    Cell, 10:549-561.-   Czauderna et al., Structural Variations and Stabilising    Modifications of Synthetic siRNAs in Mammalian Cells, Nucleic Acids    Research, 2003, Vol. 31, No. 11, 2705-2716.-   Damha, M. J., and Ogilvie, K. K. (1993) Oligoribonucleotide    synthesis. The silyl-phosphoramidite method. Methods Mol. Biol.    20:81-114.-   Donbrow (Ed.) Microcapsules and nanoparticles in medicine and    pharmacy. CRC Press, Boca Raton, Fla., pp. 125-14.-   Elbashir et al., Functional Anatomy of siRNAs for Mediating    Efficient RNAi in Drosophila melanogaster Embryo Lysate, EMBO    Journal, 2001, Vol. 20, No. 33, 6877-6888.-   Elbashir et al., RNA Interference is Mediated by 21- and    22-Nucleotide RNAs, Genes & Development, 2001, 15:188-200.-   Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K.,    and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA    interference in cultured mammalian cells. Nature, 411:494-498.-   Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA    interference is mediated by 21- and 22-nucleotide RNAs. Genes &    Dev., 15:188-200.-   Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E.,    and Mellow, C. C. (1998) Potent and specific genetic interference by    double-stranded RNA in Caenorhabditis elegans. Nature. 391:806-811.-   Graessmann, M., Michaels, G., Berg, B., and Graessmann, A. (1991)    Inhibition of SV40 gene expression by microinjected small antisense    RNA and DNA molecules. Nucleic Acids Res. 19:53-59.-   Gunnery, S., and Mathews, M. B. (1998) RNA binding and modulation of    PKR activity. Methods, 15:189-98.-   Hamada et al., Effects on RNA Interference in Gene Expression (RNAi)    in Cultured Mammalian Cells of Mismatches and the Introduction of    Chemical Modifications at the 3′-Ends of siRNAs, Antisense and    Nucleic Acid Drug Development, 2002, 12:301-309-   Hannon, RNA Interference, Nature, 2002, 418, 244-251.-   Harborth, J., Elbashir, S. M., Vandenburgh, K., Manninga, H.,    Scaringe, S. A., Weber, K., and Tuschl, T. (2003) Sequence,    chemical, and structural variation of small interfering RNAs and    short hairpin RNAs and the effect on mammalian gene silencing.    Antisense Nucleic Acid Drug Dev., 13:83-105.-   Hohjoh, J. (2002) RNA interference (RNAi) induction with various    types of synthetic oligonucleotide duplexes in cultured human cells.    FEBS Lett., 521:195-199.-   Holen, T., Amarzguioui, M., Wilger, M. T., Babaie, E., and    Prydz, H. (2002) Positional effects of short interfering RNAs    targeting the human coagulation trigger tissue factor. Nucleic Acids    Res., 30:1757-1766.-   Ji, J., Wernli, M., Klimkait, T., and Erb, P. (2003) Enhanced gene    silencing by the application of multiple specific small interfering    RNAs. FEBS Lett., 552:247-252.-   Khvorova, A., Reynolds, A., and Jayasena, S. D. (2003) Functional    siRNAs and miRNAs exhibit strand bias. Cell, 115:209-216.-   Kim, D. H., and Rossi, J. J. (2003) Coupling of RNAi-mediated target    downregulation with gene replacement. Antisense Nucleic Acid Drug    Dev., 13:151-155.-   Kim, D. H., Longo, M., Han, Y., Lundberg, P., Cantin, E., and    Rossi, J. J. (2004) Interferon induction by siRNAs and ssRNAs    synthesized by phage polymerase. Nat. Biotechnol., 22:321-325.-   Kretshmer-Kazemi Far et al., The Activity of siRNA in Mammalian    Cells is Related to Structural Target Accessibility: A Comparison    with Antisense Oligonucleotides, Nucleic Acids Research, 2003, Vol.    31, No. 15, 4417-4424.-   Liu et al., R2D2, a Bridge Between the Initiator and Effector Steps    of the Drosophila RNAi Pathway, Science, 2003, Vol. 301, 1921-1925.-   Manche, L., Green, S. R., Schmedt, C., and Mathews, M. B. (1992)    Interactions between double-stranded RNA regulators and the protein    kinase DAI. Mol. Cell. Biol. 12:5238-5248.-   Markovtsov, V., Nikolic, J. M., Goldman, J. A., Turck, C. W.,    Chou, M. Y., and Black, D. L. (2000) Cooperative assembly of an    hnRNP complex induced by a tissue specific homolog of polypyrimidine    tract binding protein. Mol. Cell Biol., 20:7463-79.-   Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and    Tuschl, T. (2002) Single-stranded antisense siRNAs guide target RNA    cleavage in RNAi. Cell, 110:563-574.-   McManus et al., Gene Silencing in Mammals by Small Interfering RNAs,    Nature Reviews Genetics, 2002, Vol. 3, 737-747.-   Melton, D. A. (1985) Injected anti-sense RNAs specifically block    messenger RNA translation in vivo. Proc. Natl. Acad. Sci. USA.    82:144-148.-   Minks, M. A., West, D. K., Benvin, S., and Baglioni, C. (1979)    Structural requirements of double-stranded RNA for the activation of    the 2′-5′-oligo(A) polymerase and protein kinase of    interferon-treated HeLa cells. J. Biol. Chem. 254:10180-10183.-   Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a    chimeric chalcone synthase gene into petunia results in reversible    co-suppression of homologous genes in trans. Plant Cell. 2:279-289.-   Ngo et al., Double-Stranded RNA Induces mRNA Degradation in    Trypanosoma brucei, Proceedings of the National Academy of Sciences    of the United States of America, 1998, Vol. 95, 14687-14692.-   Parrish, S., Fleenor, J., Xu, S., Mello, C., and Fire, A. (2000)    Functional anatomy of a dsRNA trigger: differential requirements for    the two trigger strands in RNA interference. Mol. Cell, 6:1077-1087.-   Pellino et al., R2D2 Leads the Silencing Trigger to mRNA's Death    Star, Cell, 2003, 115:132-133.-   Reynolds et al., Rational siRNA Design for RNA Interference, Nature    Biotechnology, 2004, 22, 1-5.-   Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S.,    and Khvorova, A. (2004) Rational siRNA design for RNA interference.    Nat. Biotechnol. 22:326-330.-   Romano, N., and Macino, G. (1992) Quelling: transient inactivation    of gene expression in Neurospora crassa by transformation with    homologous sequences. Mol Microbiol. 6:3343-53.-   Rossi, presentation at Integrated DNA Technologies, 2003.-   Scherer et al., Approaches for the Sequence-Specific Knockdown of    mRNA, Nature Biotechnology, 2003, Vol. 21, No. 12, 1457-1465.-   Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and    Zamore, P. D. (2003) Asymmetry in the assembly of the RNAi enzyme    complex. Cell, 115:199-208.-   Skipper, Elegant Tour de Force, Nature Reviews Genetics, 2003, Vol.    4, 79-80.-   Sledz et al., Activation of the Interferon System by    Short-Interfering RNAs, Nature Cell Biology, 2003, Vol. 5, No. 9,    834-839.-   Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and    Schreiber, R. D. (1998) How cells respond to interferons. Annu. Rev.    Biochem. 67:227-264.-   Tuschl, T., Zamore, P. D., Lehmann, R, Bartel, D. P., and    Sharp, P. A. (1999) Targeted mRNA degradation by double-stranded RNA    in vitro. Genes & Dev., 13:3191-3197.-   Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki,    H., Juni, A., Ueda, R., and Saigo, K. (2004) Guidelines for the    selection of highly effective siRNA sequences for mammalian and    chick RNA interference. Nucleic Acids Res., 32:936-948.-   Waterhouse et al., Exploring Plant Genomes by RNA-Induced Gene    Silencing, Nature Reviews Genetics, 2003, Vol. 4, 29-38.-   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D.,    Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and    Usman, N. (1995) Synthesis, deprotection, analysis and purification    of RNA and ribozymes. Nucleic Acids Res., 23:2677-84.-   Xu et al., Effective Small Interfering RNAs and Phosphorothioate    Antisense DNAs Have Different Preferences for Target Sites in the    Luciferase mRNAs, Biochemical and Biophysical Research    Communications, 2003, 306, 712-717.-   Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000)    RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA    at 21 to 23 nucleotide intervals. Cell, 101:25-33.

What is claimed is:
 1. A formulation comprising an isolated doublestranded nucleic acid, wherein said isolated double stranded nucleicacid comprises a first oligonucleotide strand comprising ribonucleotidesand having a 5′ terminus and a 3′ terminus and a second oligonucleotidestrand comprising ribonucleotides and having a 5′ terminus and a 3′terminus and wherein said double stranded nucleic acid comprises bluntends, wherein each of said first and said second strands consists of thesame number of nucleotide residues and is 25-30 nucleotides, wherein theultimate and penultimate residues of said 3′ terminus of said firststrand and the ultimate and penultimate residues of said 5′ terminus ofsaid second strand form one or two mismatched base pairs, and whereinsaid second oligonucleotide strand is sufficiently complementary to atarget mRNA along at least 19 nucleotides of said second oligonucleotidestrand length to reduce target gene expression when said double strandednucleic acid is introduced into a mammalian cell, wherein said doublestranded nucleic acid is present in an amount effective to reduce targetgene expression when said double stranded nucleic acid is introducedinto a mammalian cell.
 2. The formulation of claim 1, wherein each ofsaid first and said second strands has a length which is at least 26 andat most 30 nucleotides.
 3. The formulation of claim 1, wherein each ofsaid first and said second strands has a length of 27 nucleotides. 4.The formulation of claim 1, wherein said isolated double strandednucleic acid is a double stranded RNA and wherein said double strandedRNA is cleaved endogenously in said cell by Dicer.
 5. The formulation ofclaim 4, wherein the cleavage facilitates incorporation of the secondoligonucleotide strand into RISC.
 6. The formulation of claim 1, whereinsaid formulation is for a route of administration selected from thegroup consisting of intravenous injection, intramuscular injection,intraperitoneal injection, infusion, subcutaneous injection,transdermal, aerosol, rectal, vaginal, topical, oral, and inhaleddelivery.
 7. The formulation of claim 1, wherein said effective amountis selected from the group consisting of 1 nanomolar or less, 200picomolar or less, and 50 picomolar or less in the environment of saidmammalian cell.
 8. The formulation of claim 1, wherein said effectiveamount is administered to a mammalian subject in need thereof, andwherein said effective amount is a dosage selected from the groupconsisting of 1 microgram to 5 milligrams per kilogram of said mammaliansubject per day, 100 micrograms to 0.5 milligrams per kilogram of saidmammalian subject per day, 0.001 to 0.25 milligrams per kilogram of saidmammalian subject per day, 0.01 to 20 micrograms per kilogram of saidmammalian subject per day, 0.01 to 10 micrograms per kilogram of saidmammalian subject per day, 0.10 to 5 micrograms per kilogram of saidmammalian subject per day, and 0.10 to 2.5 micrograms per kilogram ofsaid mammalian subject per day.